In order to determine the Prevalance of Mycoplasma isolated from chicken, A total number of  200 samples collected from  birds showing  respiratory manifestations and apparently healthy chicken of  different ages(two weeks: two months) from different localities(Al-ismailia and Sharkeya Governorates). These samples include 110 samples from respiratory organs(trachea -lung -air sac) ,75 swabs from nasal cleft and 15 samples from  fluid of swollen joints. A trail for isolation and identification of different Mycoplasma was done using conventional and recent techniques. Primary isolation of the microorganism on PPLO medium, which appeared as fried egg when examined by dissecting microscope yielded118 positive samples with a total incidence rate 59%. The highest recovery rate was from respiratory organs (72.7%) followed by swabs from nasal claft (46.7%)  and samples from swollen joints (20%). Application of Digitonin test for differentiation between Mycoplasma and Acholeplasma . Mycoplasma is digitonin positive while Acholeplasma is digitonin negative. The incidence of Mycoplasma is 81.3% and the incidence of Acholeplasma is 18.6%. Biochemical characterization of the obtained isolates gave 58 isolates suspected to be M. gallispectum from different sites of isolation with percentage of 49% and 18 isolates suspected to be M.gallinarium with percentage of 15.3% and 3 isolates suspected to be M.synoviae with percentage of 2.5% and 7 isolates suspected to be M.arginini with percentage of 5.9%. Serological identification of isolates using specific antisera was applied which confirmed the presence of  M. gallispectum and M.synoviae but not other genera (M.gallinarium and M.arginini) because of  the lack of specific antisera . The minimal inhibitory concentration (MIC) results cleared that the antimicrobials (Doxycycline was followed by Erythromycin and Tilmicosin) were highly active in inhibition of  Mycoplasma  in vitro, whereas Streptomycin and Lincospectin and Ciprofloxacin were less effective against the tested isolates.